The initial step in many molecular biology procedures involves the analytical separation of DNA fragments. As is known to those skilled in the art, general steps of nucleic acid purification include cell lysis, which disrupts the cellular structure to create a lysate; inactivation of cellular nucleases, such as deoxyribonuclease (DNase) and Ribonuclease (RNase); and separation of desired nucleic acids from cell debris.
As is understood in the art, a mixture of phenol:chloroform:isoamyl alcohol (about 25:24:1) is used for denaturing proteins, and inhibiting the aforesaid RNase activity. Proteins, lipids, carbohydrates and cell debris are removed through extraction of the aqueous phase with the organic mixture of phenol and chloroform. A biphasic emulsion is formed when phenol and chloroform are added. By centrifugation of the aforementioned emulsion, the hydrophobic layer settled at the bottom and the hydrophilic layer on top. The upper phase, which contains DNA, is collected, and the DNA could be precipitated from the supernatant by adding ethanol or isopropanol in 2:1 or 1:1 ratios and high concentrations of salt. Then, the DNA precipitate is collected by centrifugation, and the salt is rinsed with ethanol.
The DNA pellet is then dissolved within a Tris-Ethylenediaminetetraacetic (TE) buffer or sterile distilled water, where Ethylenediaminetetraacetic is also termed EDTA.
For plant extraction, grinding the sample after freezing it in liquid nitrogen is necessary to break down cell wall material of the sample and allow accessing the nucleic acid, while harmful cellular enzymes and biochemicals remain inactivated. After grinding the sample, it can be re-suspended in a suitable buffer, such as cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS). This method also uses organic solvents and alcohol precipitation in later steps. Insoluble particles are removed through centrifugation to purify the nucleic acid. Soluble proteins and other materials are first mixed with chloroform, and then are separated by centrifugation. Nucleic acid must be precipitated after this from the supernatant, and washed thoroughly to remove contaminating salts. The purified nucleic acid is then resuspended, and stored in a TE (Tris-EDTA) buffer, preferably at −20° C., or in sterile distilled water.
The conventional methods described hereinabove require repeated centrifugation steps, followed by removal of supernatants, depending on the type of specimen and additional mechanical treatments, which is time consuming.
As is known to a person skilled in the art, saturated solutions of phenol are used in the conventional methods of DNA extraction and purification described hereinabove. Phenol is a flammable, corrosive and toxic carbolic acid, and, therefore, using its saturated solution is unfavorable, as it is harmful to both humans and environment. Thus, phenol and substituted phenols have been included as priority pollutants in the list of the European Union (EU) and US Environmental Protection Agency (EPA).
There is, therefore, a present need for improved methodologies and techniques for the extraction and purification of DNA.
It is, therefore, an object of the present invention to provide an alternative extraction agent for DNA extraction and purification, which facilitates a faster and safer extraction process.
These and many other objects are met in various embodiments of the present invention, offering significant advantages over the known prior art.